The invention relates to novel stabilized oligonucleotides in which at least one non-terminal pyrimidine nucleoside is modified, and to their use as a diagnostic or pharmaceutical for the treatment of viral infections, cancer or diseases in which integrins or cell-cell adhesion receptors are active.
Antisense oligonucleotides (AO) and triple-helix-forming oligonucleotides (TFO) have proved to be specific gene expression inhibitors in a large number of systems, both in vitro and in vivo [Uhlmann and Peyman, Chem. Rev. 1990, 90, 543; Milligan et al., J. Med. Chem. 1993, 36, 1923; Stein and Cheng, Science 1993, 261, 1004].
One of the main problems when using naturally occurring phosphodiesters (PO) oligonucleotides is their rapid degradation by a range of nucleolytic activities both in cells and in the cell culture medium. A range of chemical modifications was used to stabilize oligonucleotides. A review of the prior art is given, for example, by Milligan et al., supra, and Uhlmann and Peyman, supra. Stabilization against nucleolytic degradation can be effected by modifying or replacing the phosphate bridge, the sugar unit, the nucleic base, or by replacing the sugar-phosphate backbone of the oligonucleotides. Since the phosphate bridge is the center of nucleolytic attack, a large number of modifications of the internucleoside bridge were described, in particular. The most frequently used nuclease-resistant internucleoside bridges are phosphorothioate (PS), methylphosphonate (MeP) and phosphorodithioate (PA) bridges.
It must be borne in mind that the introduction of modifications alters not only the stability to nucleases, but simultaneously a large number of characteristics of the antisense oligonucleotides or triple-helix-forming oligonucleotides, such as, for example, their ability to enter cells, activation of RNase H, their specificity and their ability to hybridize with RNA (in the case of AO) or DNA (in the case of TFO) and the like. Moreover, there are indications that the stability of the serum, which is frequently used as a criterion for stability to nucleases, does not always reflect the intracellular activity [P. D. Cook in xe2x80x9cAntisense Research and Applicationsxe2x80x9d, Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chapter 9, pp. 149 et sec.]. This is why, in addition to the resistance to nucleases, the biological activity of antisense oligonucleotides or triple-helix-forming oligonucleotides gives information about the quality of such modifications.
As regards the question of the positions in the oligonucleotide at which such modifications are ideally to be effected, the following strategies have been developed [P. D. Cook (supra); Uhlmann and Peyman (supra); Milligan et al. (supra)]:
I) Exchange of All Internucleoside Bridges, for Example to Produce All-PS Oligonucleotides
This exchange gives oligonucleotides which are extremely stable to nucleases. For example, degradation by endo-nucleases (S1 nucleases) and by endo/exo nuclease P1 in an all-PS oligonucleotide is slowed down by a factor of 2-45 relative to a PO oligonucleotide [Stein et al., Nucl. Acids Res. 1988, 16, 1763]. All-PS oligonucleotides are also resistant in intact cells. In Xenopus oocytes or embryos, the degradation of microinjected PO oligonucleotides proceeds with a half-life of 30 minutes, while all-PS oligonucleotides have a half-life of over three hours under the same conditions [Woolf et al., Nucl. Acids Res. 1990, 18, 1763]. All-MeP oligonucleotides are also extremely resistant to nucleases.
The disadvantage of all-PS, or all-MeP, oligonucleotides compared with the PO oligonucleotides is that their capability of forming stable hybrids with the target RNA is reduced. A further disadvantage of the all-PS oligonucleotides are the unspecific (xe2x80x9cnon-antisensexe2x80x9d) effects, which are frequently observed in this class of compounds [Milligan et al., supra; Stein and Cheng, supra].
Other uniformly modified derivatives for example all-2xe2x80x2-O-methyl-derivatives or all-xcex1-2xe2x80x2-deoxyribo derivatives, are generally also characterized by having lost the capability of activating RNase H.
II) Copolymers of Modified and Unmodified Phosphodiester Bridges
Ghosh et al. [Anti-Cancer Drug Design 1993, 8, 15] describe a phosphorothioate-phosphodiester oligonucleotide containing various percentages of PS bridges. Their construction follows, for example, the pattern xe2x80x94(PSxe2x80x94POxe2x80x94POxe2x80x94PO)n, (PSxe2x80x94POxe2x80x94PO)n, (PSxe2x80x94PO)n, ((PO)2xe2x80x94(PS)2)n, or (POxe2x80x94PSxe2x80x94PS)n. More specifically, Ghosh et al. disclose the following constructions: (PSxe2x80x94POxe2x80x94POxe2x80x94PO)n and ((PO)2xe2x80x94(PS)2)n, wherein n=4, and (PSxe2x80x94PO)n and (POxe2x80x94PS)n wherein n=8. They teach that a PS bridge content of at least 50% is required for selective translation inhibition and that the activity drops drastically when this content is less. The present invention shows that these results are incorrect and that a PS bond content of far less than 50% is sufficient for selective inhibition if the modifications are positioned correctly (see below). Ghosh et al., furthermore teach that good results are achieved using the end capping/gap technique described under III.
The alternating exchange of every other internucleoside bridge, for example for MeP bridges (Furdan et al., Nucl. Acids Res. 1989, 17, 9193), brings no advantage in comparison with uniformly modified MeP oligonucleotides.
For example, alternatingly MeP-modified oligonucleotides equally do not activate RNase H. A comparison has shown that oligonucleotides with alternating phosphate-O-ethyl or phosphate-O-isopropyl esters and alternating MeP oligonucleotides are also less active than all-MeP or all-PS oligonucleotides [Marcus-Secura et al., Nucl. Acids Res. 1987, 15, 5749].
III) The exchange of one, two or three internucleoside bridges on the 5xe2x80x2 or the 3xe2x80x2 end of the oligonucleotides (end capping) and the exchange of one, two or three internucleoside bridges on the 5xe2x80x2 and the 3xe2x80x2 end of the oligonucleotides (gap technique).
As regards the efficacy of end capping, the results are contradictory in some cases. In particular 3xe2x80x2 end capping by means of PS, PA or MeP bridges is described as a protection against nucleases [P. D. Cook, supra, Milligan et al., supra]. A protection by means of 3xe2x80x2 end capping was also achieved by a series of other modifications. 3xe2x80x2-3xe2x80x2 end capping was described by various authors as a protection against nucleolytic degradation [Shaw et al., Nucl. Acids Res. 1991, 19, 747; Seliger et al., Mucleoside and Nucleotides 1991, 10, 469]. A further variant of 3xe2x80x2 end capping is the introduction of conjugate molecules on the 3xe2x80x2 end, which also increases stability to nucleases, such as, for example, 3xe2x80x2-dodecanol or 3xe2x80x2-acridine [P. D. Cook, supra], or 3-amino-2,3-propanediol [WO92/20697]. The gap technique, ie. the exchange of one, two or three internucleoside bridges on the 5xe2x80x2 and the 3xe2x80x2 end of the oligonucleotides, has proved particularly advantageous since, apart from PS oligonucleotides, most uniform modifications entail a lose of the capability to activate RNase H and thus a severe loss of activity. Again, a wide range of derivatives, modified phosphodiester bridges, modified sugars, modified bases, such as, for example, MeP-, PS-, PA-, 2xe2x80x2-O-alkyl- or 2xe2x80x2-F-derivatized oligonucleotides, were employed for stabilization purposes. These results are compiled in P. D. Cook supra. Within the gap, a sequence of two to four PO bonds will then suffice to activate RNase H.
Giles et al. [Anti-cancer Drug Design 1993, 8, 33] describe chimeric methylphosphonate-phosphodiester oligonucleotides in which the gap of unmodified PO bridges was reduced continuously from eight to two bridges. While a tendency was found that a reduced gap improved uptake into the cell, the oligonucleotides were not examined for their antisense activity.
An interesting comparison between various strategies can be found in Hoke et al. [Nucl. Acids Res. 1991, 19, 5743]. The authors compare the activity of a range of PS-modified antisense oligonucleotides against HSV-1 in cell culture. Their findings confirm that 3xe2x80x2, or 3xe2x80x2+5xe2x80x2, end-capped oligonucleotides (the first three internucleoside bridges being modified in each case) in the serum, similarly to all-PS oligonucleotides, are protected sufficiently against degradation by nuclease. In contrast internally modified (3 PS bridges) oligonucleotides and oligonucleotides in which only the 5xe2x80x2 end has been capped (again, the first three internucleoside bridges being modified) are degraded rapidly. In contrast, the authors found that neither 5xe2x80x2 nor 3xe2x80x2 end capping nor both are sufficient for activity within the cell, and they drew the conclusion that a uniform modification (all-PS) is required to achieve sufficient stability to nucleases in cells.
Surprisingly, it has now been found that pyrimidine nucleosides are the weak points in oligonucleotides when it comes to resistance to nucleases. If these sites are now protected by modifications which increase resistance to nucleases, this, in turn, results in a considerably improved stability and activity.
The invention therefore relates to oligonucleotides of the formula
in which at least one non-terminal pyrimidine nucleoside is modified.
Preferred oligonucleotides are those in which 2-10, in particular 3-6, non-terminal pyrimidine nucleosides are modified, and in which, especially, not more than 8 subsequent nucleotides should be modified. Particular preferred oligonucleotides are those in which additionally the 5xe2x80x2 and/or 3xe2x80x2 ends are modified, in particular those in which the first 1-5, in particular 1-3, especially 2-3, nucleotides are linked on the 5xe2x80x2 and/or 3xe2x80x2 end, preferably by phosphorothioate bridges, phosphorodithioate bridges and/or methylphosphonate bridges. Especially preferred are those modified oligonucleotides which contain one or more groups of at least 1-4, in particular 3-4, unmodified nucleotides which are linked to each other. For example, Table 1 shows the antisense oligonucleotide 01 against HSV-1, which is doubly capped with PS on the 5xe2x80x2 and 3xe2x80x2 end and which is active at a concentration of 27 xcexcM. The introduction of three PS bridges on the 5xe2x80x2 and 3xe2x80x2 end increases the activity to 9 xcexcM, the same effect being achieved by introducing a further individual PS bridge 3xe2x80x2 to a cytosine radical C (antisense oligonucleotide No. 03). The introduction of two PS bridges 5xe2x80x2 or 3xe2x80x2 to T and C (antisense oligonucleotide No. 05) or the introduction of four PS bridges 5xe2x80x2 or 3xe2x80x2 to T and C (antisense oligonucleotide No. 06) results in further increases in activity of MIC values (minimum inhibitory concentration) of 3 and 1 xcexcM, respectively. The MIC value of the corresponding all-PS derivative is also 1 xcexcm. This means that it was possible to achieve an increased stability and activity by protecting the pyrimidine nucleosides which was comparable to the all-modified oligonucleotide, but without having to suffer the above-described disadvantages of such a drastic change.
The stabilization on the pyrimidine positions as well as on the 5xe2x80x2 and/or 3xe2x80x2 ends, independently of one another, can also be effected as follows:
a) Replacement of the 3xe2x80x2 and/or the 5xe2x80x2 phosphodiester bridge, for example by a phosphorothioate, phosphorodithioate, NR1R2-phosphoramidate, boranophosphate, phosphate-(C1-C21)-O-alkyl ester, phosphate-[(C6-C12)aryl-(C1-C21)-O-alkyl] ester, 2,2,2-trichlorodimethylethyl phosphonate, (C1-C8)-alkyl phosphonate or (C6-C12)-aryl phosphonate bridge. The replacement by a phosphorothioate, phosphorodithioate, NR1R2-phosphoramidate, phosphate-O-methyl ester, phosphate-O-ethyl ester, phosphate-O-isopropyl ester, methyl phosphonate or phenyl phosphonate bridge is preferred. The replacement by a phosphorothioate, phosphorodithioate or methylphosphonate bridge is particularly preferred. The replacement by a phosphorothioate bridge is very particularly preferred.
R1 and R2 independently of one another are hydrogen or C1-C18-alkyl, C6-C20-aryl, (C6-C14)-aryl-(C1-C8)-alkyl, xe2x80x94(CH2)cxe2x80x94[NH(CH2)c]dxe2x80x94NR3R3 in which c is an integer from 2 to 6 and d is an integer from 0 to 6, and R3 radicals independently of one another are hydrogen, C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl; R1 and R2 are preferably hydrogen, C1-C8-alkyl or methoxyethyl, particularly preferably hydrogen, C1-C4-alkyl or methoxyethyl. R1 and R2 together with the nitrogen atom to which they are attached can also form a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom from the series consisting of O, S and N.
b) Replacement of the 3xe2x80x2 or the 5xe2x80x2 phosphodiester bridge by dephospho bridges [see, for example, Uhlmann and Peyman in xe2x80x9cMethods in Molecular Biologyxe2x80x9d, Vol. 20: xe2x80x9cProtocols for Oligonucleotides and Analogsxe2x80x9d, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, 355 et sec.], for example by formacetal, 3xe2x80x2-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo, dimethylene sulfone or silyl groups. The replacement by formacetals and 3xe2x80x2-thioformacetals is preferred.
c) Replacement of the sugar-phosphate backbone, for example by morpholinonucleoside oligomers [E. P. Stirchak et al., Nucleic Acids Res. 17 (1989) 6129].
d) Replacement of the xcex2-D-2xe2x80x2-deoxyribose, for example by xcex1-D-2xe2x80x2-deoxyribose, L-2xe2x80x2-deoxyribose, 2xe2x80x2-F-2xe2x80x2-deoxyribose, 2xe2x80x2-O-(C1-C6)alkyl-ribose, 2xe2x80x2-O-(C2-C6)alkenyl-ribose, 2xe2x80x2-NH2-2xe2x80x2-deoxyribose, xcex2-D-xylofuranose, xcex1-arabinofuranose, 2,4-dideoxy-xcex2-D-erythro-hexo-pyranose, and carbocyclic [for example Froehler, J. Am. Chem. Soc. 1992, 114, 8320] and open-chain sugar analogs [for example Vandendriessche et al., Tetrahedron 1993, 49, 7223] or bicyclo sugar analogs [for example M. Tarkov et al., Helv. Chim. Acta 1993, 76, 481]. The replacement by 2xe2x80x2-F-2xe2x80x2deoxyribose, 2xe2x80x2-O-(C1-C6)alkyl-ribose, 2xe2x80x2-O-(C2-C6)alkenyl-ribose or 2xe2x80x2-NH2-2xe2x80x2-deoxyribose is preferred. The replacement by 2xe2x80x2-F-2xe2x80x2-deoxyribose, 2xe2x80x2-O-(C1-C4)alkyl-ribose or 2xe2x80x2-O-(C2-C4)alkenyl-ribose or 2xe2x80x2-NH2-2xe2x80x2-deoxyribose is particularly preferred. The replacement by 2xe2x80x2-O-methyl-, 2xe2x80x2-O-allyl- or 2xe2x80x2-O-butyl ribose is very particularly preferred.
e) Replacement of the natural nucleoside bases, for example by 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5-(C1-C6)-alkyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(C1-C6)-alkylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil or 5-bromocytosine. The replacement by 5-(C1-C6)-alkyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(C1-C6)-alkylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil or 5-bromocytosine is preferred. The replacement by 5-(C3-C6)-alkyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(C1-C6)-alkylcytosine, 5-(C2-C6)-alkenylcytosine or 5-(C2-C6)-alkylcytosine is particularly preferred. The replacement by 5-pentynylcytosine, 5-hexynylurasil or 5-hexynylcytosine is very particularly preferred.
Amongst the abovementioned modifications, specially preferred modifications are those of groups a), b), c) and d), especially groups a) and d), in particular group a).
In addition, the oligonucleotides according to the invention can be linked (conjugated), for example on the 3xe2x80x2 and/or 5xe2x80x2 end, with molecules which have an enhancing effect on the characteristics of antisense oligonucleotides or of triple-helix-forming oligonucleotides (such as, for example, cell penetration, degradation by nuclease, affinity to the target RNA/DNA, pharmaco-kinetics). Examples are conjugates with poly-lysine, with intercalaters such as pyrene, acridine, phenazine, phenanthridine, with fluorescent compounds such as fluorescein, with crosslinkers such as psoralens, azido-proflavin, and with lipophilic molecules such as C12-C20-alkyl, or with derivatives thereof, such as, for example, hexamethylenetetraamine, with terpenes such as farnesol or phytol, with lipids such as 1,2-dihexadecyl-rac-glycerol, with steroids such as gallic acid, cholesterol or testosterone, with vitamins such as vitamin E, with poly- or oligoethylene glycol, with (C12-C18)-alkyl phosphate-diesters or with xe2x80x94Oxe2x80x94CH2xe2x80x94CH(OH)xe2x80x94Oxe2x80x94(C12-C18)-alkyl. Conjugates with lipophilic molecules such as C12-C20-alkyl, with steroids such as cholesterol or testosterone, with poly- or oligoethylene glycol, with vitamin E, with intercalaters such as pyrene, with (C14-C18)-alkyl phosphate diesters or with xe2x80x94Oxe2x80x94CH2xe2x80x94CH(OH)xe2x80x94Oxe2x80x94(C12-C16)-alkyl are preferred.
The preparation of such oligonucleotide conjugates is known to a person skilled in the art (see, for example, Uhlmann and Peyman, Chem. Rev. 1990, 90, 543; M. Manoharan in Antisense Research and Applications, Crooke and Lebleu, Eds. CRC Press, Boca Raton, 1993, Chapter 17, pp. 303 et seq., EP0552766A2).
Moreover, the oligonucleotides according to the invention can carry 3xe2x80x2-3xe2x80x2 and 5xe2x80x2-5xe2x80x2 inversions [described, for example, in M. Koga et al., J. Org. Chem. 56 (1991) 3757] on the 3xe2x80x2 and/or the 5xe2x80x2 end.
The invention furthermore relates to processes for the preparation of the compounds according to the invention by processes known to a person skilled in the art, in particular chemical synthesis, to the use of the compounds according to the invention for the preparation of a pharmaceutical, and to a process for the preparation of a pharmaceutical which comprises mixing the oligonucleotides according to the invention with a physiologically acceptable excipient and, if appropriate, suitable additives and/or auxiliaries.
Quite generally, the present invention also extends to the use of therapeutically active oligonucleotides for the preparation of a pharmaceutical in which at least one non-terminal pyridine nucleoside is modified. Therapeutically active oligonucleotides are generally to be understood as meaning antisense oligonucleotides, triple-helix-forming oligonucleotides, aptamers (RNA or DNA molecules which can bind to specific target molecules, for example proteins or receptors (for example L. C. Bock et al., Nature 1992, 355, 564) or ribozymes (catalytic RNA, see, for example, Castanetto et al., Critical Rev. Eukar. Gene Expr. 1992, 2, 331), in particular antisense oligonucleotides.
Moreover, the present invention furthermore relates to the use of oligonucleotides having at least one non-terminal and modified pyrimidine nucleoside as a diagnostic, for example for detecting the presence or absence or the amount of a specific double-stranded or single-stranded nucleic acid molecule in a biological sample.
For their use according to the invention, the oligonucleotides have a length of approximately 6-100, preferably approximately 10-40, in particular approximately 12-25, nucleotides. Again, the above-described preferred ranges, modifications and conjugations apply.
The pharmaceuticals of the present invention can be used for example for the treatment of diseases caused by viruses, for example by HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.
Examples of antisense oligonucleotides according to the invention which are active against such targets are:
a) against HIV, for example
The pharmaceuticals of the present invention are also suitable, for example, for the treatment of cancer. For example, oligonucleotide sequences can be used which are directed against targets responsible for the formation or growth of cancer. Examples of such targets are:
1) Nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb, c-fos c-fos/jun, PCNA, p.120
2) Cytoplasmic/membrane-associated oncoproteins such as, for example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl
3) Cellular receptors such as, for example, EGF receptor, c-erbA, retinoid receptors, protein-kinase-regulatory subunit, c-fms
4) Cytokins, growth factors, extracellular matrix such as, for example, CSF-1, IL-6, IL-1a, IL-1b, IL-2, IL-4, bFGF, myeloblastin, fibronectin.
Antisense oligonucleotides according to the invention which are active against such targets are, for example,
a) against c-Ha-ras, for example,
c) c-myc, for example,
d) c-myb, for example,
e) c-fos, for example,
f) p120, for example,
g) EGF receptor, for example,
h) p 53 tumor suppressor, for example,
j) Antisense oligonucleotide against cdc2 kinase:
5xe2x80x2-G*T*C*TTC*CAT*AGT*TAC*T*C*A-3xe2x80x2 (XXI)xe2x80x83xe2x80x83(SEQ ID NO: 22)
k) Antisense oligonucleotide against PCNA (proliferating cell nuclear antigen):
5xe2x80x2-G*A*T*CAGG*CG*TGC*CTC*A*A*A-3xe2x80x2 (XXII)xe2x80x83xe2x80x83(SEQ ID NO: 23)
l) Antisense oligonucleotide against IGF-1:
5xe2x80x2-T*G*A*AGA*CGAC*A*TGAT*G*T*G-3xe2x80x2 (XXIII)xe2x80x83xe2x80x83(SEQ ID NO: 24)
m) Antisense oligonucleotide against bFGF translation start site:
5xe2x80x2-G*G*C*TGC*CA*TGGT*C*C*C-3xe2x80x2
5xe2x80x2-farnesyl-G*G*C*TGC*CA*TGGT*C*C*C-3xe2x80x2
5xe2x80x2-phytyl-G*G*C*TGC*CA*TGGT*C*C*C-3xe2x80x2
5xe2x80x2-hexadecyl-G*G*C*TGC*CA*TGGT*C*C*C-3xe2x80x2
5xe2x80x2-cholesteryl-G*G*C*TGC*CA*TGGT*C*C*C-3xe2x80x2
5xe2x80x2-hexamethylenetetraamine-G*G*C*TGC*CA*TGGT*C*C*C-3xe2x80x2
5xe2x80x2-G*G*C*TGC*CA*TGGT*C*C*C-hexadecyl-3xe2x80x2
5xe2x80x2-G*G*C*TGC*CA*TGGT*C*C*C-cholesteryl-3xe2x80x2
5xe2x80x2-G*G*C*TGC*CA*TGGT*C*C*C-vitamin E-3xe2x80x2
5xe2x80x2-G*G*C*TGC*CA*TGGT*C*C*C-bile acid-3xe2x80x2 (XXIV)xe2x80x83xe2x80x83(SEQ ID NO: 25)
n) Antisense oligonucleotide against bFGF codon 58 ff
5xe2x80x2-C*T*G*TAGT*T*TGAC*G*TGT*G*G*G-3xe2x80x2 (XXV)xe2x80x83xe2x80x83(SEQ ID NO: 26)
o) Antisense oligonucleotide against FGF receptor:
5xe2x80x2-G*G*C*CCC*T*CCAGC*CC*CACAT*C*C*C-3xe2x80x2 (XXVI)xe2x80x83xe2x80x83(SEQ ID NO: 27)
Furthermore, the pharmaceuticals of the present invention are suitable, for example, for treatment of diseases affected by integrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2, ICAM or ELAM.
Antisense oligonucleotides according to the invention which are active against such targets are, for example,
a) VLA-4, for example,
b) ICAM, for example,
c) ELAM-1, for example,
The pharmaceuticals can be used for example in the form of drug preparations which can be administered orally, for example in the form of tablets, coated tablets, hard- or soft-gelatin capsules, solutions, emulsions or suspensions. They can also be administered rectally, for example in the form of suppositories, or parenterally, for example in the form of injectable solutions. For the preparation of drug preparations, these compounds can be incorporated into therapeutically inert organic and inorganic excipients. Examples of such excipients for tablets, coated tablets and hard-gelatin capsules are lactose, corn starch or derivatives thereof, tallow and stearic acid or salts thereof. Suitable excipients for the preparation of solutions are water, polyols, sucrose, invert sugars and glucose. Suitable excipients for injectable solutions are water, alcohols, polyols, glycerol and vegetable oils. Suitable excipients for suppositories are vegetable and hardened oils, waxes, fats and semi-liquid polyols. The drug preparations can also comprise preservatives, solvents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts for regulating the osmotic pressure, buffers, coating agents, antioxidants and, if appropriate, other therapeutic active substances. A preferred form of administration is an injection. To this end, the antisense oligonucleotides are formulated in a liquid solution, preferably in a physiologically acceptable buffer such as, for example, Hank""s solution or Ringer""s solution. However, the antisense oligonucleotides can also be formulated in solid form and dissolved or suspended prior to use. The doses preferred for systematic administration are approximately 0.01 mg to approximately 50 mg per kg of body weight per day.